We propose to determine the structural requirements of the formylatable and nonformylatable E. coli methionine tRNAs (f-tRNA Met and m-tRNA Met) for recognition by E. coli methionine tRNA synthetase and for attachment of methionine. In addition we propose to determine the structural requirements for recognition of f-tRNA Met by E. coli methionine tRNA transformylase and initiation factor IF2 and the structural basis for the failure of these proteins to recognize m-tRNA Met. Finally, we propose to determine the structural requirements for recognition of m-tRNA Met by the aminocyl-tRNA binding protein, T factor, and the structural basis for the failure of this protein to recognize f-tRNA Met. The approach which we will use to determine which nucleotide sequences are required for the attachment of methionine to f-tRNA Met and m-tRNA Met and for the subsequent formylation of met-f-tRNA Met involves three steps: (a) chemical modification of purified tRNA, (b) separation of the tRNA after modification into components which are active and inactive with respect to the biological activity being examined, and (c) analysis of the structure of the active and inactive molecules for the sites of the modifications. The structural requirements for recognition of the two nucleic acids by the four proteins involved in protein synthesis will be studied by fractionating chemically modified tRNAs on the basis of their ability to form specific complexes with these proteins and determining the sites of modifications which alter binding affinity. The solution conformation of f-tRNA Met, m-tRNA Met, met-f-tRNA Met Met-m-tRNA Met, and fMet-f-tRNA Met will be studied by chemical and physical methods in order to investigate the role of ordered structure in specific recognition by the proteins and to study conformational changes in the nucleic acids following acylation, formylation or specific protein-nucleic acid complex formation.